Immunology & Microbiology 10 : Apoptosis, Necrosis & NETosis

What are some of the causes of cell injury?

- Infectious agents - Chemical agents - Oxygen deprivation - Physical agents (heat, radiation, trauma) - Genetic defects - Immune cell activation (CTL, ADCC) - Aging (cellular senescence)

What are the signs of REVERSIBLE cell injury?

1) Blebbing membrane 2) Swelling of the ER, clumping of nuclear chromatin and mitochondrial swelling 3) Aggregation of intramembranous particles 4) Autophagy

Describe the terminal apoptotic changes

1) Caspase activation initiates multiple proteolytic cascades within the cell. 2) Extensive protein cross-linking by transglutaminases leads to cytoskeletal changes and formation of apoptotic bodies 3) Chromatin breakdown into 180-200bp intra-nucleosomal DNA fragments 4) DNA fragmentation caused by enzyme CAD (caspase activated DNase) - shown by DNA laddering assays

Describe the extrinsic apoptotic Fas-FasL mechanism

1) Fas ligand (e.g. T cells) binds to Fas at peripheral cell surfaces 2) Binding of FasL causes a conformational change in Fas, which then binds death-domain containing adaptor proteins 3) Adaptor proteins recruit and activate caspase 8, which cleaves caspase 3 4) Activated caspase 3 cleaves I-CAD, freeing CAD (caspase-activated DNase) to enter nucleus and cleave DNA

What are the stages of inflammasome activation during pyroptosis?

1) PRIMING - Microorganism recognition by NLRP3 causes inflammasome to assemble - Assembles NLRP3, ASC adaptor & procaspase-1 2) ACTIVATION - CARD domains on NLRP3 and procaspase interact - Causes cleavage from procaspase to caspase-1 - Caspase-1 cleaves Pro-IL-18 & Pro-IL-1beta into IL-18/IL-1Beta - These are released through a pore formed by GSDMD (also activated by the caspase-1) - The ILs cause the inflammatory death

What are the signs of IRREVERSIBLE cell injury/Necrosis?

1) Rupture of lysosomes and autolysis 2) Pyknosis (nuclear shrinkage), karyolysis (nuclear fading due to chromatin loss) or karyorrhexis (nuclear fragmentation - looks basophilic) 3) Lysis of ER and mitochondrial swelling Necrosis is a sequence of morphological changes that follow cell death following irreversible injury. - Caused by enzymatic hydrolysis of cell components, and protein denaturation - E.g. liquefactive or caseous (TB)

Give 2 examples of diseases causing apoptosis

1) Salmonella can activate caspases and cause apoptosis 2) Huntington's leads to neuronal death via genetic changes that cause apoptosis

What two other things events to coincide after Fas-FasL interactions for apoptosis to become irreversible?

1) Specific protein adaptors that recognise death domain proteins must be present 2) Changes in mitochondrial permeability (+ integration signalling by BCL-2 family members). Regulators/Inhibitors present which determine if execution caspases are produced (e..g BCL-2 inhibits and BAX promotes). If yes, leads to DNA destruction

What are the immune-modulatory functions of NETs?

1) They can prime other immune cells to induce sterile inflammation or potentiate autoimmunity by stimulating IFN responses owing to NET-associated oxidized DNA and antimicrobial peptides. 2) NETs can also occlude the vasculature by promoting thrombosis and obstruct important organ areas, they can capture metastatic tumours and delay wound healing in diabetes.

What are the biochemical changes of cell injury?

ATP depletion-> rapid shutdown of homeostatic pathways Generation of Reactive Oxygen Species (ROS)-> activates multiple signalling pathways, kinases, phosphatases, NF-kB, phospholipases Changes in membrane permeablility-> breakdown of concentration gradients of ions and metabolites Loss of calcium homeostasis Mitochondrial damage.

What is autophagy?

AUTOPHAGY = self eating. It is a cell survival mechanism that aims to derive energy by eliminating defective organelles via lysosomes Rarely does it lead to cell death, often only a temporary response to cell injury, It can be triggered in starvation and can delay or prevent programmed cell death. It is shown schematically in image: 1) In response to starvation/hypoxia, formation of isolation membrane (P13K & Beclin 1), which wrap up defective organelles. 2) LC3 complex helps form the autophagosome, which then fuses with a lysosome to form an autolysosome 3) Defective organelles then broken down to provide ATP and precursors for protein synthesis.

What is the control station called which decides whether execution caspases will go on to cause apoptosis

BCl-2 rheostat. Loss of the rheostat function causes inhibition of apoptosis (seen in cancers e.g. B cell lymphoma)

What is cellular senescence?

Cellular ageing is caused by progressive accumulation of sublethal injuries that may lead eventually to loss of function or cell death. Senescent cells accumulate free radical damage, abnormally folded proteins and AGE modified proteins. AGE = Advanced Glycation Endproducts Senescence is a permanent arrest of cell division cycle. Behaviour of ageing cells been referred to as senescence-associated secretory phenotype (SASP)

How do immune privileged tissue avoid immune response?

EXPRESS FAS-LIGAND Tissue express Fas ligand, which binds T cell Fas and causes apoptosis E.g. in eye, consequence is that corneal transplants can be done across MHC boundaries without any immune rejection. Another consequence seen in cancer cells e.g. melanomas, which act as if immune privilege, by expressing FasLigand

What are the intrinsic and extrinsic signals for apoptosis?

INTRINSIC - Cytochrome C - Developmentally programmed - Lack of specific growth factor - Unrepaired DNA damage EXTRINSIC - TNF-a - TNFR interaction - FAS ligand - FAS interaction - Involved death receptors - Cytotoxic T cell granule release

What is apoptosis?

It is also known as programmed cell death, and is a normal component of development and health of multicellular organisms Cells die in response to a variety of stimuli and during apoptosis they do so in a controlled, regulated fashion. Apoptosis is a process in which cells play an active role in their own death (which is why apoptosis is often referred to as cell suicide). This makes apoptosis distinct from necrosis in which uncontrolled cell death leads to lysis of cells and initiation of inflammatory responses

Describe how mitochondrial damage results in cell injury

Mitochondrial disfunction: any change e.g. increased calcium, can affect mitochondrion + change to changes in permeability (Mitochondrial permeability transition/MPT) Signs of MPT = leaking of cytochrome C from mitochondrion to cytoplasm Leakage of other molecules e.g. protons, elating to breakdown of proton-motor force and acidification of the cytosol

What triggers NET formation?

NETosis is induced in neutrophils response to microbial cues and endogenous danger signals e.g. innate immune receptors acting through intracellular mediators that include reactive oxygen species (ROS) produced by NADPH oxidase

What are the key enzymes involved in DNA repair that caspases attack?

PARP - The ability of the enzyme PARP (poly (ADP-ribose) polymerase) to repair DNA damage is prevented following cleavage of PARP by caspase-3. DNA topoisomerase II is a nuclear enzyme essential for DNA replication and repair. Caspases can inactivate this enzyme leading to DNA damage. Lamins are intra-nuclear proteins that maintain the shape of the nucleus and mediate interactions between chromatin and the nuclear membrane. Degradation of lamins by caspase 6 results in chromatin condensation and nuclear fragmentation.

What is pyroptosis?

Pyroptosis is carried out in highly self-programmed way that involved innate immune cells and activation of inflammasomes It is NOT caused by physical tissue injury

What are the two outcomes of a sub-cellular response to damaged proteins?

REPAIR: Chaperones bind to and refold it to form a normal protein again. DEGRADATION: If repair can't occur, proteins and ubiquitinated and degraded by proteasomes.

How are apoptotic cells recognised? How is this different to necrotic cells?

Recognised by scavenger receptors and others e.g. PS receptors/CD14. Leads to changes in macrophages gene expression. Causes secretion of anti-inflammatory cytokines (TNF-beta) and decrease in inflammatory cytokines (IL-1beta). This is because DAMPs remain hidden. Phagocytosis occurs, leading to no inflammatory response For necrotic cells, released DAMPs mean macrophages release pro-inflammatory cytokines

Describe slow NETosis and non-lytic NETosis (neutrophil extracellular traps)

SLOW NETOSIS: - Nuclear delobulation & Disassembly of nuclear envelope - Cellular depolarisation & Chromatin decondensation - Plasma membrane rupture & release of NETs after intracellular assembly NON-LYTIC NETOSIS - Rapid degranulation & expulsion of chromatin - Extracellular assembly of NETs

Describe the extrinsic apoptotic TNF-a mechanism

When macrophages are activated, they can release TNF as a soluble molecule through TACE (TNF-a converting enzyme) Target cell has two types of TNFa receptor (p55/p75) TNF-alpha receptors have the following structure: - TNFR1 has trimeric structure that interacts with TNFa on outside of membrane - Internal side contains death domains, and TRADD (TRF receptor associated death domain) - Leads to activation of apopttic pathway (caspases) through signalling.